IL-6 is the standardized name of a cytokine called B lymphocyte differentiating factor, interferon .beta..sub.2, 26 Kd protein, hybridoma/plasmacytoma growth factor, hepatocyte stimulating factor, etc.
IL-6 induces activated B cells to be differentiated into antibody forming cells. For T cells, IL-6 induces T cells stimulated by mitogens to produce IL-2 and induces the expression of IL-2 receptor on a certain T cell line or thymocytes. For blood forming cells, IL-6 induces the growth of blood forming stem cells synergistically in the presence of IL-3. Furthermore, recently, it was reported that IL-6 acted like thrombopoietin. As stated here, IL-6 has a variety of biological activities and is expected to be useful for clinical application.
IL-6 is produced by various cells. It is produced by lymphocytes and is also produced by human fibroblasts stimulated by Poly (I).Poly (C) and cycloheximide (Eur. J. Biochem., 159, 625, (1986)). Murine IL-6 is produced in mouse cells, which are stimulated by Poly (A).Poly (U) (Immunopharmacology, 21, p. 33, (1991)). Inducers for stimulation are diverse, and those include known cytokines such as IL-1, TNF and IFN-.beta., growth factors such as PDGF and TGF-.beta., LPS, PMA, PHA, cholera toxin, etc. (Science, 235, 731 (1987)). Moreover, it is reported that human vascular endothelial cells, macrophages, human glioblastomas, etc. also produce IL-6 (Immunol.,142,144, (1989), J. Immunol., 141, 1529, (1988), Japanese Patent Laid-Open No. 88-296688)). Furthermore, it is also known that the productivity can be further enhanced by stimulating cells using an inducer and subsequently treating the cells by a metabolic inhibitor such as verapamil, cycloheximide or actinomycin D, etc. (J. Immunol., 144, 4242-4248 (1990)). However, how IL-6 is different in activity, structure, etc. due to the differences in producing cells, inducing substance, etc. is unknown. To effectively utilize IL-6 as a medicine at least, it is necessary to develop a mass production system and to clarify the physical properties and biological activities of produced IL-6. However, no process for efficient mass production of high-quality IL-6 containing sugar chains useful for clinical application has been established yet.
It is known that IL-6 can be purified by letting CPG (controlled pore glass) adsorb it in batch operation, recovering it by an acid and using a polyclonal antibody column, gel permeation chromatography, ion exchange chromatography and reverse phase high performance liquid chromatography using a C1 column in combination (Eur. J. Biochem., 168, 543 (1987)) or by using membrane concentration, gel filtration chromatography, dialysis, ion exchange chromatography, FPLC and reverse phase high performance liquid chromatography (HPLC) in combination (Proc. Natl. Acad, Sci. USA, 82, 5490 (1985)). Recombinant IL-6 derived from Escherichia coli is purified by using urea treatment, dialysis, guanidine hydrochlorate treatment, gel filtration chromatography and ion exchange chromatography in combination (Tosoh Research Report, Vol. 32, No. 2 (1988)). Furthermore, human BCDF produced by Escherichia coli can be purified to a protein purity of 99% or more and an endotoxin content of 0.6 EU/mg protein or less, by two-stage reverse phase HPLC using chemical bond type (C.sub.8) silica gel as a packing material(Japanese Patent Laid-Open No. 90-186996). However, these conventional purification processes are complicated and not suitable for mass processing for industrial production. In addition, reverse phase HPLC conditions are not mild for proteins, and are liable to cause denaturation or association of proteins, being not adequate for obtaining high quality IL-6 product.
As described above, the industrial utilization of IL-6 as a medicine is possible only after successful mass production of high quality IL-6 product and the clarification of its properties. The present invention solves these problems and provides a process to produce glycosylated IL-6 compositions in high quality under the control of good manufacturing process.